As a Medical Oncology fellow at the Fred Hutchinson Cancer Research Center I have been impressed by the toxicities associated with conditioning regimens used for conventional hematopoietic stem cell transplantation (HSCT). The recent development of nonablative conditioning has lowered HSCT-associated toxicities allowing for treatment of older patients and of patients with comorbidities, who would not have been eligible for conventional HSCT. Most nonablative conditioning regimens contain irradiation, which is thought to be required to prevent graft rejection. However, low doses of irradiation are associated with increased risks of late malignancies, and avoiding these late toxicities would be highly desirable for patients treated for non-malignant diseases (e.g. sickle cell disease, immunodeficiencies) and pediatric patients with malignant diseases. We hypothesize that other less toxic preparative strategies can be developed to further reduce the irradiation dose yet ensure stable donor hematopoietic cell engraftment. In theory, this can be achieved by a) increasing the recipient's immunologic "visibility" resulting in increased graft-versus-host and graft-versus-tumor activities, and/or b) decreasing the donor's immunologic "visibility" resulting in decreased donor-directed immune reactions that cause rejection. In the dog leukocyte antigen identical littermate HSCT model, as in human patients, long-term stable engraftment can be achieved with irradiation doses of 200 cGy when used in conjunction with potent post-grafting immunosuppression. In preliminary canine studies, however, irradiation doses of 50 cGy used with cyclosporine given for 5 weeks resulted in only transient mixed donor-host chimerism. We propose to use these preliminary studies as a baseline to develop strategies to achieve stable engraftment in this model. In Specific Aim 1, host dendritic cells (DC) will be expanded before transplant using FIt-3-ligand to enhance host-directed immune responses for more efficient eradication of graft-rejection-mediating T cells in the recipient. DC-expansion and function will then be correlated with engraftment. In Specific Aim 2, donor-specific tolerance will be induced in this model by testing 4 strategies. These strategies include blockade of the CD28/B7, CD40/CD154, and CD27/CD70 costimulatory pathways, and sequential exposure of host T cells to donor-specific antigen and the antimetabolite methotrexate to trigger activation-induced cell death in donor-reactive T cells. Effective strategies will then be combined in Specific Aim 3 to achieve synergistic effects. The results of these studies will help devise non-toxic transplant regimens that do not rely on general immunosuppression of the recipient, but rather specifically target immune processes that mediate graft-rejection and graft-versus-host disease.